CORE UNIT B: ANATOMY Principal Investigator: Allan I. Basbaum, Ph.D. (Anatomy) ; Key Personnel: TBN Justification for the Anatomy Core: In response to the Study Section's critique of the somewhat unusual organization proposed for the Scientific Core, we have made significant changes, moving to a more traditional structure. The home of the Scientific Core will be under the direction of Dr. Allan Basbaum and it will be located within his laboratory. The great advantage of this arrangement, of course, is that Dr. Basbaum is a highly experienced neuroanatomist and will be in a position to oversee the technicians who will service the Scientific Core as well as to provide expertise to the three Pi's. Most importantly, we envision that the Scientific Core will serve as much more than a place where day-to-day histological work is performed. There are four major features of light microscopic neuroanatomy: 1) determination of the most appropriate techniques to be applied and development of the protocols; 2) tissue preparation (cutting, staining, etc); 3) tissue observation (regular fluorescence and confocal microscopy); and 4) analysis of the histological tissue. The Scientific Core is designed to provide excellent support in each of these areas. To this end, we propose to have three key personnel associated with the Scientific Core. Dr. Basbaum will oversee the Scientific Core. He will interact with the Pi's and will coordinate the work of the two research technicians in the Scientific Core laboratory, Ms. Sandy Canchola (who worked for many years in the laboratory of Dr. Henry J. (Peter) Ralston and Mr. Larry Ackerman, who comes to us from the laboratory of Lily and Yuh Nung Jan (See below). All necessary equipment for tissue preparation, immunostaining, in situ hybridization, etc. as well as tissue observation and documentation are available in the Dr. Basbaum's laboratory, which is now located in the Rock Hall building at the new UCSF Mission Bay Campus. Dr. Basbaum's laboratory has a large, dedicated section for histology. A portion of the histology space will be assigned for the Scientific Core, to be used by students and fellows of the Pi's, as well as by the histologist, Ms. Sandy Canchola. The students and fellows will work under the guidance of Ms. Canchola and Dr. Basbaum. We are only requesting a minimal supply budget for the Scientific Core facility, for basic reagents and other supplies. All the more costly reagents (e.g. antibodies) will be acquired from individual components' budgets. Storage space for supplies for individual projects will be available in the Basbaum lab, which has considerable space in its -80[unreadable]C freezers and 4[unreadable]C refrigerators. The shared antibody resource will also be physically located in the Basbaum laboratory, providing important reagents to all Pis. With respect to the use of confocal microscopes, all Pis have access to state-ofthe- art confocal microscopes. Drs. Basbaum and Levine are on a funded multiuser equipment grant that has recently purchased a two-photon confocal that can image living cells, Dr. Messing has a confocal available to him at the Gallo Center and Dr. von Zastrow has access to two confocal microscopes in Genentech Hall, where his laboratory is located at the Mission Bay campus. Equipment is a small part of what is required to do first-rate confocal microscopy. Experimental design, tissue preparation and understanding the strengths and limitations of confocal microscopy are key. This is where the Scientific Core brings great strength to the overall project. Dr. Basbaum not only has considerable experience using confocal microscopy, having published numerous papers using this approach, but his laboratory has hired a superb, highly experienced technician, who can only be described as an expert in this area. Larry Ackerman worked for over 20 years in the Jan laboratory, but recently left as the direction of their laboratory changed considerably. Dr. Basbaum was fortunate to hire Mr. Ackerman. Because it is not essential to have such an experienced individual working full time on this proposal, (much of the work involves tissue preparation), we propose to bring Mr. Ackerman on 25% time, which we believe is sufficient to address the needs of the group. Mr. Ackerman's strength is in teaching individuals in the laboratory to use the confocal microscope so as to take advantage of its strengths. He will also be a resource for design of experiments and most importantly for the adaptation of new technologies as they appear. One excellent example is the use of quantum dots for highresolution light microscopic analysis. Mr. Ackerman has the experience, as illustrated by his inclusion on many of the Jan papers, to add incredible value to the group. We do not see Mr. Ackerman as someone who will do the work; rather he will coordinate the efforts of and teach the students and fellows, discussing the plans with Dr. Basbaum and the individual Pi's. We believe that this is an extremely appropriate way to bring expertise to the projects, without requiring a very large financial commitment. Fortunately, Dr. Basbaum and Mr. Ackerman bring expertise that is highly relevant to several of the questions being asked in the course of the studies proposed by Drs. Levine, Messing and von Zastrow. For example, Dr. Levine will be studying drug-induced translocation of PKCe. The approaches required to complete these studies are very familiar to Dr. Basbaum who has not only studied PKCy translocation (at both the light and electron microscopic levels) but who has also'Studied internalization of the neurokinin-1 (NK1) receptor in response to environmental stimuli and exogenous drugs. The methods required to recognize and document translocation of immunoreactive products are very familiar to Dr. Basbaum, who will consult in the course of these experiments. Mr. Ackerman similarly brings incredible expertise with confocal microscopy, which is essential for such experiments to be performed reliably and with high sensitivity. The Core wi|l similarly provide excellent support for the studies proposed in component #3 by Dr. von Zastrow. These studies involve monitoring opioid and pYadrenergic receptor localization and trafficking in nociceptive neurons. Dr. Basbaum has extensive experience in localization of endogenously expressed opioid receptors in neural tissue, in both rat and mouse, and in fact, has published in vivo studies on the internalization of the mu opioid receptor in spinal cord. His experience with the plethora of opioid receptor antibodies that are available (many of which are of no value) will be particularly helpful. Furthermore, Dr. Basbaum will provide critical instruction in methods to define the cellular distribution in nociceptive neuron subpopulations. Finally, given the remarkable association of the delta opioid receptor with substance P, the fact that Dr. Basbaum's laboratory generated a preprotachykinin-A mutant mouse will prove very valuable to Dr. von Zastrow. Not only will the mice be readily available but also Dr. Basbaum has considerable experience in examining the distribution of a variety of G protein linked receptors in this mouse. The organization of the Scientific Core will require structure so that it is efficient. All projects have need for the Scientific Core, but it is difficult to anticipate ahead of time the exact percentage effort for the use of this Scientific Core by each project. As noted by the review group, the Pis of this PPG proposal have a long track record of working together, which we fully expect will include being able to accommodate each other's needs for the personnel in the Scientific Core. Furthermore, given the decreased number of projects, we expect that the Scientific Core personnel will have adequate time to complete all the proposed projects. We propose that Pi's will make initial requests to Dr. Basbaum for help with particular projects. Depending on the nature of the project, Dr. Basbaum will assign either Ms. Canchola, or Mr. Ackerman to the project. If Ms. Canchola will be working independently on the project (See below), then she will obtain tissue as required (after perfusion of animals in the Pi's laboratory), and perform traditional light microscopic staining methods, including immunocytochemistry using DAB protocols as well as immunofluorescence. As required, Ms. Canchola will also run in situ hybridization protocols, including probe generation and hybridization. Considerable double labeling work will be performed, as illustrated in some of Dr. Messing's proposed studies in the dorsal root ganglion. This is another area very familiar to Dr. Basbaum. A good example is exemplified by his studies of the 5HT3 receptor, which involved a number of double labeling light microscopic approaches, including double immunofluorescence as well as combined in situ hybridization and immunocytochemistry. Dr. Basbaum's laboratory also regularly uses tyramide signal amplification (TSA), which will prove very valuable to some of the proposed studies, including the GRK and beta-adrenergic receptor studies described by Drs. Levine and von Zastrow. The expected low levels of beta-adrenergic receptors will likely require some amplification protocol, such as TSA, for their detection. About 50% of Ms. Canchola's work in the Scientific Core laboratory will be assigned to independent projects that originate in the different PI laboratories. The remaining time will be performed in association with students and fellows from the laboratories of thee Pis. With respect to training students, Dr. Basbaum strongly believes in the policy of see one, help with one, do one. In other words, the goal is to train the different students and fellows so that they are comfortable working independently within the Scientific Core laboratory, under the guidance of Ms. Canchola, and with the availability of Dr. Basbaum for more complicated questions where the technology interfaces more directly with the scientific question.